The protozoan parasite Toxoplasma gondii is a ubiquitous human pathogen[unreadable] which has emerged as a leading opportunistic infection associated with[unreadable] AIDS. Clinical management of toxoplasmosis has traditionally relied on[unreadable] antifolates, but complications associated with the chronic therapy needed[unreadable] for immunodeficient patients have left us with no adequate treatment for[unreadable] this devastating disease. This proposal seeks to employ newly developed[unreadable] genetic and pharmacological tools to investigate drug sensitivity and[unreadable] resistance in Toxoplasma, focusing particularly on folate metabolism and[unreadable] the parasite's bifunctional dihydrofolate reductase-thymidylate synthase[unreadable] enzyme (DHFR-TS). Goals of this research include the development of[unreadable] improved treatment strategies for acute toxoplasmosis.[unreadable] Sequences and probes derived from the T. gondii DHFR-TS gene have been[unreadable] employed to examine the predicted structure of the enzyme in wild-type[unreadable] parasites, and to develop functional vectors for transient and stable[unreadable] molecular transformation of the parasite. Clinical cases of antifolate-[unreadable] resistant toxoplasmosis and similar mutants isolated in the lab will be[unreadable] screened for differences in sensitivity to combined[unreadable] pyrimethamine/sulfonamide treatment (in addition to sensitivity to[unreadable] pyrimethamine or sulfa alone), and examined for possible DHFR-TS mutations[unreadable] or altered gene expression. Mechanisms of DHFR-independent resistance will[unreadable] be identified by genetic means. DHFR-TS enzyme function will be assessed[unreadable] in transgenic parasites bearing mutations derived from four sources:[unreadable] naturally occurring allelic variation, mutations identified from drug-[unreadable] resistant laboratory strains and clinical isolates, modeling studies on[unreadable] the T. gondii enzyme, and point mutations thought to be associated with[unreadable] antifolate resistance in field isolates of the related parasite Plasmodium[unreadable] falciparum (malaria). Findings from this research will be combined with[unreadable] structure/function studies on the recombinant DHFR-TS enzyme, to assist in[unreadable] the design of novel antifolates with improved activity against the[unreadable] parasite.[unreadable] Because of the tremendous power of genetic techniques for the[unreadable] identification and analysis of drug targets, available transformation[unreadable] schemes will be modified to permit: (1) Targeted gene disruption and[unreadable] replacement, used in this context to examine mutations at the DHFR-TS[unreadable] locus; (2) Insertional mutagenesis and marker rescue, used to clone[unreadable] potential targets for therapeutic intervention and negative selectable[unreadable] markers for gene replacement studies; (3) Molecular cloning by[unreadable] complementation, focusing on DHFR-independent antifolate resistance genes[unreadable] which appear to be particularly important for pyrimethamine resistance in[unreadable] Toxoplasma; and (4) Optimal expression and overexpression of recombinant[unreadable] protein. In addition to their value for the analysis of folate metabolism[unreadable] and drug resistance, these tools should be broadly applicable to other[unreadable] studies on the biology and biochemistry of Toxoplasma.[unreadable]